Spinal cord injury (SCI) is often followed by the development of debilitating spasms in the muscles innervated from the spinal cord below the site of injury. More than 80% of individuals with SCI have spasms and spasticity that significantly disrupt residual motor function, cause debilitating pain, and interrupt sleep. Treatment of spasms with conventional antispastic drugs (e.g., baclofen) is often not adequate, or not tolerated because of adverse side effects such as lethargy and weakness. Serotonin (5-HT), the brain stem-derived neurotransmitter, serves a critical role in tuning the excitability of the spinal motoneurons by facilitating persistent calcium currents (Ca2+ PICs) that amplify and prolong responses to synaptic input. When SCI eliminates this major source of 5-HT, spinal motoneurons are initially left in a depressed state and are not able to produce adequate muscle contractions. Over the weeks after injury, however, Ca2+ PICs in spinal motoneurons spontaneously recover, helping with restoration of rudimentary motor functions, but also contributing to spasms. Recently, Dr. David Bennett and his colleagues showed that the recovery of Ca2+ PICs is afforded by a molecular mechanism called RNA editing. Months after SCI, there are alterations in mRNA editing of one of the serotonin receptors (2C receptor or 5-HT2CR), which lead to increased expression of the 5-HT2CR isoforms that are active without 5-HT. Such constitutive receptor activity coincides with a restoration of large Ca2+ PICs in the motoneurons of the injured rats. Editing is catalyzed by specific enzymes-adenosine deaminases that act on RNA (ADAR1 and ADAR2), whose regulation is poorly understood. Most of them encode receptors or channels (e.g., 5-HT2CR, ionotropic glutamate receptors, Kv1.1 potassium and Cav1 calcium channels) that are expressed in the neuronal cells throughout the brain and spinal cord. Editing of these molecules modulates their Ca2+ permeability and kinetics, thus influencing intrinsic excitability of neurons. In our preliminary studies we found that SCI-induced 5-HT2CR editing changes are related to downregulation of ADAR2 (but not ADAR1) mRNA expression in SCI vs. control animals. We, therefore, hypothesize that, in addition to 5-HT2CR, SCI triggers editing changes in other receptors and channels whose mRNA is edited by ADAR2, which collectively contribute to the recovery of motorneuron excitability and the concurring development of spasticity. We also hypothesize that downregulation of ADAR2 is mediated by specific regulatory pathways that can be identified by screening the global transcriptional response to SCI using genome-wide sequencing and novel bioinformatics tools. To test these hypotheses, we propose the following Aims:
Specific Aim 1 : To investigate if RNA editing of AMPA and kainate glutamate receptors, voltage gated potassium channel Kv1.1, and/or L-type calcium channel Cav1 is altered in the spinal cord of SCI animals.
Specific Aim 2 : To identify networks, pathways, and individual molecules that regulate downregulation of ADAR2 following SCI. We will use genome-wide transcriptome sequencing technology (RNA-Seq) and weighted gene coexpression network analysis (WGCNA) in order to investigate ADAR2 transcriptional control and its alteration following SCI.
Specific Aim 3 : To identify SCI-induced alterations in editing and ADAR2-related regulation that are specific for motoneurons. While studies in Aims 1 and 2 will be performed in whole spinal cord preparation, the experiments proposed in this Aim will use laser microdissected spinal motoneurons. To summarize, the proposed research will identify ADAR2 substrates whose editing is altered by SCI as well as identify regulators of ADAR2 expression and function. These studies will advance our understanding of the neuropathology of SCI and spasticity. Most importantly, they will suggest possible targets for the development of novel antispastic drug therapy.
In the United States, the incidence of spinal cord injury (SCI) is significantly higher among members of the Military and Veterans (429 per million) compared to civilians (25-59 per million). Spasticity is a common sequella of SCI and is a major complaint of injured individuals. The existing antispastic agents are marked by low efficacy and severe side effects. The development of new therapeutic strategies that overcome these limitations would greatly improve the quality of life among Veterans with SCI. Our preliminary data suggest that SCI triggers alterations in the efficiency of the unique molecular mechanism called RNA editing. This leads to a host of RNA editing changes that contribute to the development of spasticity. The proposed studies aim to characterize these RNA editing-related mechanisms, thus providing a framework for the development of novel treatments.